Method of sterilizing



Patented Apr. 6, 1937 2,075,845 METHOD OF STERILIZING Paul M. Gross and Lawrence F. Dixon, Durham,

., assignors to Liggett 8t Myers Tobacco Company, New York, N. Y., a corporation of New Jersey No Drawing. Application May 5, 1933, Serial 8 Claims.

This invention relates to a new and useful process of sterilizing various substances or materials to free them of bacterial or micro-organism contamination, and is based upon the discovery made by us after extensive research work and actual demonstrations, that ethylene oxide, either in the form of a gas or otherwise, when employed in the presence of the proper quantity of moisture, will effectually kill or reduce the 10 number of living bacteria or micro-organisms with which a substance or material is contaminated.

An important object of the invention is to provide a process that may be practically carried out in a simple manner and without requiring the employment of expensive or complicated apparatus; an ordinary gas-tight box, tank or receptacle of proper dimensions having a suitable loading and unloading door and an inlet tor the sterilizing medium, being all that is required in most instances, although the process may be successfully carried out in some cases without the employment of such apparatus.

Among the numerous advantages incident to our new process of sterilizing or reducing the bacterial or micro-organism count of contam-' inated substances or materials, we cite the following:

1. The method can be applied to objects, materials, or substances without the necessity of altering them by the application of liquids, solutions or solids, by merely causing the gasto come in contact with them in the presence of the proper amount of moisture for predetermined periods of time.

2. The method or process can be carried out at ordinary temperatures and involves no supplementary heating process or treatment. This is of great importance as many sterilizing processes require heat which fundamentally alters or even may damage the materials being sterilized.

3. The sterilizing agent when in form of a gas makes it possible to sterilize inaccessible porticns of the object, material or substance without immersing or inserting it in, or washing it with, a liquid or solution as is commonly necessary with other sterilizing agents or processes.

4. The processor method permits of the sterilization or reduction of the bacterial or microorganism contamination of objects, materials or substances within closed packages or containers (provided the containers are not gas tight) since the gas used can penetrate to the interior of the package or container.

5. It is also possible with this process to sterilize materials or substances packed or filled in gas-tight containers by filling the containers with the contents in them with the gas ethylene oxide together with the necessary amount of moisture, then sealing the container gas tight and allowing the gas to remain in contact with the material in the sealed container until opened prior to use.

In practicing the invention we prefer to em ploy a gas-tight box, tank or enclosure of suitable dimensions, depending upon the character or the quantity of material or substance to be treated and which enclosure has a loading and unloading opening adapted to be closed substantially air-tight by a door or other closure, and said box, tank or other enclosure, which. in some instances may be a closed room, has a pipe leading thereto through which the ethylene oxide gas may be admitted. and a second pipe or conduit leading to a suitable evacuating apparatus to exhaust air from the enclosure.

The substance or material to be treated is introduced into the enclosure and the door then closed. Preferably the air is then exhausted from the enclosure and ethylene oxide gas introduced therein until a predetermined pressure is reached, the pressure depending somewhat upon the character of the contamination, the concentration of the gas, and the duration of the treatment, which to be effective, must be carried out in the presence of a proper quantity or percentage of moisture present in the material or substance being treated or introduced into the enclosure.

In order to enable others skilled in the art to understand and practice the invention, we will now proceed to describe the same by giving several concrete examples of the manner in which we have actually practiced the method in the treatment of. certain materials, or substances, and upon different types of micro-organisms; but we do not wish to be understood as limiting or restricting ourselves to the use or application of the process to the particular substances or 4r materials mentioned, or to the particular proportions or quantities of ethylene oxide used, or to the particular mode of application, as it will be perfectly apparent from the detailed description to follow, that variations or modifications 50 of the process will suggest themselves to those skilled in the art and may be made, without departing from thespirit of the invention:

A sample of raisins which contained a sufficient quantity of moisture was divided into three equal iii portions. On the first portion a bacteria and microorganism count was made and the sample or raisins as purchased was found to contain 1,863 bacteria or microorganisms per gram. The second and third samples were each placed in sterile containers which were then evacuated until the pressure within the container was 740 mm. below atmospheric. Ethylene oxide was then admitted to one of these containers until the pressure within it was atmospheric. The container was then cut off from the source of ethylene oxide and allowed to stand closed for 180 minutes, at the end of which time the pressure within the container fell to 30 mm. below atmospheric pressure indicating absorption of the gas by the raisins. Air was then admitted to the container through a sterile cotton filter and the sample of raisins removed under sterile conditions and a bacteria and microorganism count made upon it using an approved bacteriological technique as before. The sample contained no bacteria or microorganisms and was therefore sterile.

The other container holding the third sample which had been evacuated as explained above was allowed to stand for the same period of time as was the first container but no ethylene oxide was added to it. Sterileair was then admitted to it as in the case of the first container and the sample which it contained was removed under sterile conditions and a count made on it. It was thus found to contain 2,070 bacteria and microorganisms per gram in substantial agreement with the first sample. This blank experiment in which all operations and steps were carried out exactly the same as with the first sample, except that of addition of ethylene oxide and which failed to produce sterilization, indicates clearly that the ethylene oxide in the presence of moisture is the effective sterilizing agent and that such sterilization is not the result of steps or operations incidental to the bringing of the ethylene oxide in contact with the substance such as evacuating, standing, etc.

As further representative of various classes of foodstuffs, a sample of breakfast cereal known as white rolled oats and a sample of chopped fresh meat both in the presence of sufficient moisture were subjected to treatment under similar conditions to those used for the raisins outlined above. Both the oats and the meat were found to be sterile after such treatment though contaminated with bacteria and microorganisms prior to the treatment with ethylene oxide.

A sample of soil was also subjected to treatment under the following conditions:

A bacteria'and microorganism count was made on the soil as removed from the ground and it was found by the approved bacteriological technique to be heavily contaminated with bacteria and microorganisms. A portion of this sample was then heated to 60 C. for one hour after which it was again examined for bacteria and microorganisms. It was found to still contain large numbers of resistant spore forming organisms which had not been killed by the heat treatment. Samples of the heated soil and of the original unheated soil were placed in separate sterile containers and subjected to the action of ethylene oxide gas for two hours. Both samples of soil contained sufficient moisture for the ethylene oxide to act effectively. The soil samples were then removed from the containers under sterile conditions and a bacteria and microorganism count made on both samples. Both samples of soil treatedwith ethylene oxide were found to be sterile. Since one of these samples had been previously heated to 60 C. and still contained resistant spore forms of bacteria and microorganisms and these were subsequently killed by'the ethylene oxide treatment this method possesses the further advantage of killing resistant spore forms of bacteria and microorganisms which are diiiicult or impossible to kill by the usual sterilizing agents and are only effectively killed by high temperature or steam autoclave sterilization.

Since highly resistant bacteria occur in sugars of the general type known as thermophilic bacteria, samples of commercial sugars were tested by an approved technique and determinations of the numbers of such organisms were made. Thus for instance a certain sample was found to contain 10 such organisms per gram. Among these were those types which when such sugars are used in canning produce such difficulties as flat sours and the like. A portion of this sample was moistened slightly with water to provide the proper moisture content necessary and subjected to the action of ethylene oxide gas fortwo and one-half hours in a concentration corresponding to 44.8 lbs. per 1000 cu. ft. or a partial pressure of the gas of 0.4 atmosphere. This treated portion was then cultured as was the original sample of sugar and was found to be sterile, containing no microorganisms, thus showing the effectiveness of this treatment in killing such thermally resistant bacteria and microorganisms.

Our discovery consists not only in the fact that ethylene oxide will sterilize materials or substances contaminated with bacteria or microorganisms but in the fact that the presence of the proper amount of moisture is essential to vi the success of such sterilization. We will therefore point out in detail and in a number of different instances, how this process of sterilization is dependent upon the presence of the proper quantity of moisture.

As a first illustration may be cited the case of the sterilization of sugar previously discussed. One sample of dry sugar which contained 1.5% moisture was treated with ethylene oxide gas in a dosage or concentration corresponding to apit proximately 44.8 lbs. per 1000 cubic feet of container space for 2.5 hours. After this treatment it still contained some microorganisms showing that it had not been sterilized under these conditions. In a second trial another sample of the same sugar which contained 1.5% moisture was treated for 2.5 hours with a much higher dosage of ethylene oxide gas; namely, one corresponding to a concentration of approximately 111 lbs. of gas to 1000 cubic feet. After this treatment the sugar still contained miscroorganisms indicating that even the increased dosage of ethylene oxide was unable to effect sterilization in the absence of sufficient moisture. A third sample of the same sugar had suflicient water added to it to raise its moisture content to approximately 10% and was then treated with the same dosage as was used on the first sample; namely, 44.8 lbs. per 1000 cubic feet for 2.5 hours. After this treatment the sugar was found to be free of bacteria or microorganisms and had been effectively sterilized.

As a second illustration we may cite the effect of moisture on the effectiveness of this sterilization process in the case of a single kind of (iii organism; namely, the spore form of Bacillus mycoides Fliigge, Smears of these spores were placed on dry sterile cotton so that the aggregate moisture content of cotton and smear was approximately 3%. The cotton was then exposed to a concentration corresponding to approximately 111 lbs. of ethylene oxide per 1000 cubic feet of container space for three hours. At the end of this time the number of the baccilli present on the cotton were too numerous to count, showing that the ethylene oxide gas had had practically no eifect in killing them. The trial was repeated using the same dosage and the same time, but with a smear of these organisms on sterile cotton which had been saturated with water. Under these conditions the cotton was found to be completely sterile after the three hour exposure.

In order to define more closely the influence of moisture, we have carried out a number of trials with a substance whose moisture content we could control through a range of values. We chose for this purpose cut tobacco in cigarettes since all parts of it were readily accessible to the gas and its moisture content could be changed over a fairly large range of values without affecting its state or texture. A large number cf such cigarettes from the same batch were divided up into separate lots and the moisture content of each lot brought to predetermined values by conditioning them in desiccators above aqueous solutions of H2304 of the proper strengths. Each lot of different moisture content was then treated with ethylene oxide gas for five hours at a con centration corresponding to 55.8 lbs. per 1000 cubic feet. The original untreated cigarettes had bacteria and microorganism counts varying from 1775 to 6000 bacteria or microorganisms per gram. The results of the counts on the treated lots were as follows: 7

Those containing 2.1% moisture had an average count of 560 per gram;

Those with a moisture content of 3.02% an average count of 560 per gram;

Also, those with 5.40% moisture an average count of 110 per gram;

Those with 8.8% moisture an average count of 50 per gram;

While those with moisture contents of 9.6% up to 14.5% (the highest moisture) were all sterile.

It is thus evident that in this case sterilization with ethylene oxide gas is effective only when the moisture content is approximately 9%.

In the light of these examples and as the result of numerous other trials we have found that sterilization of bacteria or microorganism contaminated substances or materials by the gas ethylene oxide can only be effected provided the proper amount of moisture is present. We wish to stress the fact that the presence of this moisture in proper amount is a necessary and essential part of our process, as without its presence it'is not possible to kill completely bacteria or microorganisms with which the substance or material being treated may be contaminated.

Since effective sterilization by ethylene oxide gas depends on the dosages or concentrations of gas employed as well as on the presence of the proper moisture content, we will detail here the necessary conditions of dosage and time of exposure to produce sterilization in a few instances by way of illustration. These figures or' values are not however, to be construed as limiting the process to these particular values but are cited this value or 11.1 lbs. per 1000 cubic feet an exposure of 24 hours is required for this organism. Again we have found that while a dosage of 111 lbs. per 1000 cubic feet will kill the vegetative forms of Bacillus mycoides in 10 minutes, these organisms are killed in 3 hours with a lower dosage corresponding to 33.3 lbs. per 1000 cubic feet. On the other hand a dosage of as high as 77.8 lbs. per 1000 cubic feet of ethylene oxide gas is required to kill the spore forms of this organism and an exposure time of three hours. These instances indicate that provided the dosages used are sufficiently high it is possible to vary the conditions of efiective sterilization by changing either the dosage or concentration employed or by changing the period of exposure, always however under the limiting condition that a proper and sufficient amount of moisture is present.

We are aware of the fact thatthe gas ethylene oxide has been proposed for use as an insecticide for eradicating organisms of the type of flour beetles, moths, etc.,'in stored materials, but to our knowledge it has not been used and is not known to be effective as a sterilizing or germicidal agent to kill those living forms which are classed as germs, bacteria or microorganisms. An essential feature of our discovery of the sterilizing o-r germicidal action of ethylene oxide gas is the fact that this action takes place only in the presence of a proper and suflicient quantity of moisture and without this such action does not take place effectively. Furthermore in order to be effective as a germicidal or sterilizing agent the gas ethylene oxide must be used in dosages or concentrations many times greater than needed when it is employed as an insecticide.

The above illustrations and descriptions of the conditions necessary to carry out the process properly show the effectiveness of the ethylene oxide gas in the presence of moisture as a sterilizing or germicidal agent for a wide variety of materials contaminated with various organisms. To show more fully its effectiveness as a germicidal agent for killing specific bacteria and microorganisms of various types, representative members of the.

important classes of microorganisms and bacteria, were treated by this process as follows:

Cultures of the organisms were taken from nu trient emulsions known to contain the specific organism in question and placed on balls of sterile cotton together with the proper amount of moisture and the balls put in sterile test tubes plugged of the ethylene oxide gas at a concentration corre spondingto approximately 33.4 lbs. per 1000 cubic feet of container space and for lengths of time of 2.5 or 3 or 4 hours, depending on the organism teriological technique for the organism in question;

showed ithe'abs'enoe of these organisms thus proving thatigthg otton ballshad been sterilized by treatment for the length of time indicated below for each organism concerned.

The second tube containing each of the kinds of organisms was kept the same length of time as the first tube but was not subjected to the action of the ethylene oxide. On culturing these untreated cotton balls in the same manner as the first balls all of these untreated organisms were found to be alive, thus showing that the original organisms were virile and also that the ethylene oxide was the effective agent, in the presence of moisture, in killing the organisms on the treated balls.

In order to eliminate the possibility of this treatment only causing inhibition of growth and not sterilization, the untreated and treated inoculated materials had cultures made from them after 48 hours, 1 week and 2 weeks after the treatment. Those that were untreated showed positive growth, whereas those which were, treated showed no growth in any instance, thus demonstrating that they were really sterilized and that the organisms were killed and their growth not simply inhibited.

Employing the above procedure the germicidal action of ethylene oxide gas in the presence of moisture has been shown for the following microorganisms in the groups indicated:

The concentration was equivalent to 33.4 lbs. per 1000 cubic feet of space.

The following organisms of the intestinal group were killed by an exposure of 2.5 hours: Brucella (porcine variety), Para Typhoid-A, Para-Typhoid-B, Flexner Dysentery-B. Dysentery Mt. Desert, B. proteus, B. alkaligenes, Vibrio cholera, B. cntcrz'ditfs, B. aerogenes. The following of this group were killed by a 4 hour exposure: Dysentery Shiga, B. Morganii No. 1.

The following of the upper respiratory group were killed by 2.5 hours exposure: B. influcnzae, B. diph heriac. M. catarrhalis, H emolytz'c streptococcus, Streptococcus oerz'dans, pneumococcus Type I, tubercle bacilli, B. pertussis.

In the aerobic spore bearing group of bacilli, B. subtilis was killed by an exposure of 3 hours and B. anthrax by an exposure of 2.5 hours.

In the anerobic sporebearing Bacilli group, B. welchii was killed by an exposure of 4 hours and the following by an exposure of 2.5 hours: Clostridium oedematis maligm', B. botulinus, B. tctam'z', C'lostridium histolyticum.

The following fungi were killled by an exposure of 2.5 hours: Aspergillus glaucum, Oidium, Torula, Monilia, C'occidzodes immitus, Blastomy'ces, yeast (beer), Sporothrir schenockii, trichophyton inquinale, trichophyton gypseum, trichophyton interdigitalis, Actinomyces.

The following unclassified microorganisms were Iliilled by an exposure of four hours: B. Zeptiseptihas, and B. mallei, while the following were killed by an exposure of 2.5 hours: Staphylococcus citreus, Streptococcus pyogenes, B. pyocyaneus, B. prodigiosus, B. lactis acidophilus, B. acne.

The virus which is the cause of the mosaic disease of tobacco plants and which is reported as a typical and resistant virus we have successfully killed by the use of ethylene oxide gas in the presence of the proper amount of moisture.

The above cases show the wide scope of the germicidal power of the gas ethylene oxide when employed in the presence of the proper amount of moisture and in sufficiently high dosages or concentrations.

We have furthermore found it possible to employ solutions of ethylene oxide or its homologs instead of ethylene oxide gas to sterilize materials or substances or infected objects or areas by treating them with a solution of ethylene oxide or its homologs in water or other solvents or mixtures of solvents in those cases where treatment in such manner seems advantageous or desirable.

Thus we have found that the following solutions of ethylene oxide are effective germicidal agents as shown by their ability to kill the organism Staphylococcus aureus in not more than 2 minutes; an 11% solution of ethylene oxide in ethylene glycol, a 10% solution of ethylene oxide in 95% ethyl alcohol, a 10% solution of ethylene oxide in absolute ethyl alcohol.

Solutions of ethylene oxide in such solvents though their germicidal action may not be as great as solutions of equivalent strength of certain other substances possess the great advantage that they do not have an injurious or caustic effect on the tissues even when the ethylene oxide is present in such high concentrations.

These solutions of ethylene oxide in the solvents named are merely given as illustrations of the possible uses of solutions of ethylene oxide in solvents or mixtures of solvents as germicidal agents and are not to be construed as limiting our use of ethylene oxide in solution to these cases, as we have found by numerous trials that solutions of ethylene oxide of various strengths in various solvents are effective germicidal agents for widely different types of bacteria and microorganisms. For instance we have found that the homologs of ethylene oxide, such as propylene oxide, are quite effective in accomplishing the desired results.

Because of the wide range of usefulness of the process and the great variety of materials or substances, as well as the widely difierent bacterial or microorganism contaminations that may be effectively treated, it is obviously impracticable, in a specification of this kind, to attempt to give specific examples of the exact amount of moisture, the precise concentration of the ethylene oxide gas or the time of exposure applicable to each or to all, nor do we believe this to be necessary in view of the specific examples herein given, as it will be clear from the examples given that those desiring to practice the invention may do so successfully by following the procedure described and using only ordinary care and the expected technique of one skilled in the art, suffice it to say that our work in this field seems to indicate that the effectiveness of the germicide depends upon the presence of sumcient water or moisture on the surface of the material with which the organisms are in contact, although we do not wish to be limited to this or any other theory. The factors which determine what the value of this moisture content is vary so much from one material to another due largely to the natural hydroscopicity of the different materials that it is impractical to make a general statement concerning them which will apply to all materials, suflice it to say that the moisture or water must be sufficient to react with the ethylene oxide gas to produce ethylene glycol either wholly or in part. Thus the hygro scopic character of tobacco is such that it requires approximately 9% moisture for ethylene oxide to be effective, whereas in the case of sugar this limit would. be somewhat lower, probably 6% to 8%.

Our new process is especially adapted for use in sterilizing surgical instruments, dressings, barinto ethylene glycol.

bers supplies, such as towels, brushes, razors and the like, and may be economically and effectively carried out by simply placing the various articles in an ordinary steam sterilizer of the types now in use for steam sterilization; and then introducing ethylene oxide gas into the sterilizer instead of steam, and this may be done by first evacuating the sterilizing chamber or without evacuation depending upon the conditions.

It is our belief as a result of our extensive research work in this field, that the efiectiveness of ethylene oxide as a germicide depends upon the presence of the proper amount of water or moisture in or upon the surface of the materials or substances being treated. As we have stated, it is not possible to make a general statement as to the exact amount of Water or moisture required for the various substances treated. The reasons for this are as follows:-

FirstEach substance will normally absorb or retain a particular amount of moisture dependent upon its natural hydroscopicity. The addition of water to the substances will have to be in such amount as to take care ofthis hydroscopicity and in addition leave an amount of moisture present in or upon the surface as a film which will be adequate to dissolve the ethylene oxide gas and then re-act with it, producing ethylene glycol either wholly or in part.

Second-The progress of this inter-action between ethylene glycol and water or'moisture depends upon a number of factors, prominent among which may be mentioned the temperature, the time of contact and the nature of the surface of the substance which is being treated. This last is true as it is well known that this re-a-ction is one which is catalized by the presence of other substances than the water and ethylene, oxide, namely, in this case, the surface of the material being treated.

Since ethylene oxide gas must first dissolve in water or a moisture film, and subsequently react with the moisture, an amount of moisture must be present sufiicient to convert the ethylene oxide This quantity of moisture cannot be specifically stated in the general case. However, as an example, if the surface of a material or substance was of such extent and nature that it carried .1 of a gram of moisture in or upon it as a film available for reaction, this quantity of moisture would be sufficient to convert approximately 2.4 grams of ethylene oxide into ethylene glycol.

Our improved process may also be advantageously employed in sterilizing stored surgical sutures or ligatures, as where the latter are enclosed within sealed containers of glass or the like in the presence of ethylene oxide, either in the form of a gas or in the form of a liquid.

We have herein given only a few specific examples of the materials, substances or objects introducing ethylene oxide gas into the enclosure and permitting the objects, substances or materials to remain in the enclosure and in the presence of the gas and a predetermined amount of moisture for a pre-determined length of time, the amount of added moisture being suificient to react with the gas to produce ethylene glycol.

2. The method of sterilizing to reduce bacterial or micro-organism contamination, which consists in adding moisture to and in enclosing the contaminated objects, substances, or materials within an enclosure, evacuating the enclosure, then introducing therein ethylene oxide. gas at a pre-determined concentration, and permitting the objects, substances or materials to remain in the presence of the said gas and a predetermined amount of moisture for a predetermined length of time, the amount of added moisture being suffieient to react with the gas to produce ethylene glycol.

3. The method of sterilizing microbe-contaminated objects, substances or materials, which consists in adding moisture to and in exposing the substances or materials to the action of ethylene oxide gas in the presence of a predetermined quantity of moisture, the amount of added moisture being sufficient to react with the gas produce ethylene glycol.

4. The method of sterilizing to reduce bacterial or micro-organism contamination, which consists in adding moisture to and in exposing contaminated substances or materials to the action of ethylene oxide gas in the presence of a pre-determined quantity of moisture depending upon the nature of the. substances or materials and the type of micro-organism, the amount of added moisture being sufiicient to react with the gas to produce ethylene glycol.

5. The process of freeing food stuffs, such as dried fruits, meat, sugar, or the like, of bacteria and micro-organisms, which consists in adding moisture to and in subjecting'such materials to the action of the gas ethylene oxide in the presence of a pre-determined quantity of moisture taking into account the normal moisture content of said material and for a pre-determined length of time depending upon the nature and degree of the contamination, the amount of added moisture being suflicient to react with the gas to produce ethylene glycol.

6. The process of sterilizing materials, such as surgical instruments and dressings, medical supplies and the like, which consists in placing such materials or supplies in a container and introducing therein ethylene oxide gas, together with a pre-determined amount of moisture, the amount of added moisture being sufficient to react with the gas to produce ethylene glycol.

'7. The process of freeing food stuffs, or other materials or substances of bacteria or micro-organisms, which consists in placing such materials having a pre-determined moisture content in a container, removing a part of the air from the container by evacuation adding additional moisture, then admitting the gas ethylene oxide into the container until the partial pressure of concentration or dosage of the ethylene oxide reaches a pre-determined value, and permitting the material to remain in the container for a pre-determined length of time, depending upon the nature and extent of the contamination, the amount of added moisture being suflicient to react with the gas to produce ethylene glycol.

8. The method of sterilizing or freeing soil of bacteria or micro-organisms, which consists in adding a suitable amount of moisture to the soil the soil, the amount of added moisture being suibringing ethylene oxide in the presence of a pre-- flcient to react with the gas to produce ethylene determined amount of moisture in contact with glycol. the soil for a pre-determined length of time de- PAUL M. GROSS.

5 pending upon the type of bacteria or micro-or- LAWRENCE F. DIXON. 5

ganism and the amount of moisture contained in 

